The practice of modern medicine relies heavily on effective antibiotic therapy to combat bacterial infections. In their absence, many surgeries and chemotherapeutic regimens used to combat cancer and suppress chronic inflammatory conditions would simply not be possible. Development of multidrug-resistant (MDR) bacterial strains threatens to undermine these advances and represents a major global health challenge. A common resistance mechanism in gram negative rod (GNR) bacteria is secretion of a beta-lactamase that hydrolyzes the beta-lactam ring common to all members of the penicillin class. Hundreds of diverse beta-lactamases, which vary in their catalytic mechanism and substrate specificity, have evolved in response to treatment of infections with multiple b-lactam antibiotics Carbapenems represent the last line of effective defense against MDR GNRs expressing extended spectrum beta-lactamases (ESBLs) capable of hydrolyzing all non-carbapenem beta-lactams. However, their effectiveness has been compromised by the recent emergence of plasmid-encoded carbapenemases. To effectively contain and/or prevent outbreaks of carbapenem resistant bacterial infections and to offer optimal patient care, rapid detection of carbapenemase activity in clinical specimens is imperative. We have pioneered a rapid, phenotypic method relying on mass spectrometry to detect carbapenemase activity, by selectively monitoring for the appearance of carbapenem hydrolysis products. In the first aim of this application, we will refine and optimize our current MS- based assay to detect -lactam ring hydrolysis of carbapenem antibiotics. We will define necessary kit components and assay protocols for detection of carbapenem hydrolysis activity in bacterial isolates. In the second aim of this application, we will perform a blinded clinical study using selected retrospective and prospective clinical specimens from Yale-New Haven Hospital in order to establish biologic sensitivity and specificity of the overall protocol. Future phases of development will apply the technology to primary specimens, which will be followed by multicenter clinical trials in pursuit o FDA 510(k) clearance of the final product.